Caveolae and L-type Calcium Channels in Cardiomyocytes

描述(申请人提供)：项目摘要：通过心肌细胞表面膜L型钙通道的钙内流调节多种细胞过程，包括收缩、分泌、细胞信号和基因表达。这项研究的长期目标是：1)基于相关蛋白的独特的大分子复合体，区分心肌细胞中不同的L型钙通道；2)确定这些不同的钙通道群体如何具体地促进各种钙调节的细胞过程；以及3)了解不同群体的钙通道的变化如何促进心血管疾病。这些目标最初将集中于定位于被称为小窝的特化膜微域的Cav1.2 L类型钙通道，该微域由标志性蛋白质小窝3(Cav-3)定义。Cav1.2通道在与最近发现的Cav-3突变相关的长QT综合征的发生中的作用将被确定。利用免疫沉淀技术、GST-Cav-3下拉、免疫共聚焦显微镜、免疫金电子显微镜、靶蛋白siRNA敲除、Cav1.2通道和关键相关蛋白的异源表达以及Cav-3的条件性敲除，我们将解决三个特定目标：1)确定定位于小凹的Cav1.2通道的相对丰度和组成；2)确定Cav-3和相关的核心支架蛋白是否对正常的基础L型钙电流及其(？)-AR调节是必需的；3)确定长QT相关Cav-3基因突变对CAV1.2 L型钙通道的影响。 相关性：这些研究将提供定位于小凹的钙通道蛋白亚群的分子定义，从而为长QT综合征和与心脏小窝标志性蛋白Caveolae-3突变相关的危及生命的心律失常的发生提供机械性见解。此外，这项研究将影响我们对心力衰竭和心房颤动的理解，因为在这些疾病中已经检测到小窝和钙通道的变化。最终，对小窝和L型钙通道的分子理解有可能启发这些普遍存在的心血管疾病的新疗法。

DESCRIPTION (provided by applicant): Project Summary: The influx of Ca through L-type Ca channels in surface membrane of cardiomyocytes regulates a variety of cellular processes including contraction, secretion, cell signaling, and gene expression. The long-term goals of this research are 1) to discriminate between distinct populations of L-type Ca channels in cardiomyocytes based on the unique macromolecular complex of associated proteins; 2) to determine how these different populations of channels specifically contribute to various Ca-regulated cellular processes; and 3) to understand how alterations in the different populations of Ca channels contribute to cardiovascular disease. These goals will be approached initially focusing on Cav1.2 L-type Ca channels localized to the specialized membrane microdomains known as caveolae which are defined by the signature protein caveolin-3 (Cav-3). The contribution of Cav1.2 channels to the genesis of the long QT syndrome associated with recently identified mutations in Cav-3 will be defined. Using immunoprecipitation techniques, GST-Cav-3 pull-down, immunoconfocal microscopy, immunogold electron microscopy, siRNA knockdown of targeted proteins, heterologous expression of Cav1.2 channels and key associate proteins, and conditional knockout of Cav-3 in mice, we will address three specific aims: 1) Define the relative abundance and composition of the Cav1.2 channels localized to caveolae in ventricular myocytes; 2) Determine if Cav-3 and associated core scaffolding proteins are essential for normal basal L-type Ca current and its (¿-AR regulation in ventricular myocytes; 3) Determine the impact of long QT-related Cav-3 genetic mutations on Cav1.2 L- type Ca channels. Relevance: These studies will provide molecular definition of the subpopulation of Ca channel proteins localized to caveolae and thus offer mechanistic insights into the genesis of the long QT syndrome and life-threatening arrhythmias associated with mutations in the signature protein of caveolae in the heart, caveolin-3. Furthermore, this research will impact our understanding of heart failure and atrial fibrillation given that alterations in caveolae and Ca channels have been detected in these diseases. Ultimately, a molecular understanding of caveolae and L-type Ca2+ channels has the potential to inspire new therapies for these prevalent cardiovascular diseases.